Polypeptides

ABSTRACT

The present invention relates to polypeptides, and nucleic acids DNA encoding these polypeptides, capable of eliciting an immune reaction against cancer, methods for generating T lymphocytes capable of recognising and destroying tumour cells, and pharmaceutical compositions for the treatment, prophylaxis or diagnosis of cancer.

[0001] The present invention relates to polypeptides, and nucleic acidsDNA encoding these polypeptides, capable of eliciting an immune reactionagainst cancer, methods for generating T lymphocytes capable ofrecognising and destroying tumour cells, and pharmaceutical compositionsfor the treatment, prophylaxis or diagnosis of cancer.

[0002] Cancer develops through a multistep process involving severalmutational events. These mutations result in altered expression/functionof genes belonging to two categories: oncogenes and tumour suppressorgenes. Oncogenes arise in nature from proto-oncogenes through pointmutations or translocations, thereby resulting in a transformed state ofthe cell harbouring the mutation. Oncogenes code for and functionthrough a protein. Proto-oncogenes are normal genes of the cell whichhave the potential of becoming oncogenes. In the majority of cases,proto-oncogenes have been shown to be components of signal transductionpathways. Oncogenes act in a dominant fashion. Tumour-suppressor geneson the other hand, act in a recessive fashion, i.e. through loss offunction, and contribute to oncogenesis when both alleles encoding thefunctional protein have been altered to produce non-functional geneproducts.

[0003] In the field of human cancer immunology, the last two decadeshave seen intensive efforts to characterise genuine cancer specificantigens. In particular, effort has been devoted to the analysis ofantibodies to human tumour antigens. The prior art suggests that suchantibodies can be used for diagnostic and therapeutic purposes, forinstance in connection with an anti-cancer agent. However, antibodiescan only bind to tumour antigens that are exposed on the surface oftumour cells. For this reason, the effort to produce a cancer treatmentbased on the immune system of the body has been less successful thananticipated.

[0004] A fundamental feature of the immune system is that it candistinguish self from nonself molecules and that it does not normallyreact against self molecules. It has been shown that rejection oftissues or organs grafted from other individuals is an immune responseto the foreign antigens on the surface of the grafted cells. The immuneresponse comprises a humeral response, mediated by antibodies, and acellular response. Antibodies are produced and secreted by Blymphocytes, and typically recognise free antigen in nativeconformation. They can therefore potentially recognise almost any siteexposed on the antigen surface. In contrast to antibodies, T cells,which mediate the cellular arm of the immune response, recogniseantigens only in the context of major histocompatability complex (MHC)molecules, and only after appropriate antigen processing. This antigenprocessing usually consists of proteolytic fragmentation of the protein,resulting in polypeptides that fit into the groove of the MHC molecules.This enables T cells to also recognise polypeptides derived fromintracellular protein fragments/antigens.

[0005] T cells can recognise aberrant polypeptides derived from anywherein the tumour cell, in the context of MHC molecules on the surface ofthe tumour cell. The T cells can subsequently be activated to eliminatethe tumour cell harbouring the aberrant polypeptide. In experimentalmodels involving murine tumours it has been shown that point mutationsin intracellular “self” proteins may give rise to tumour rejectionantigens, consisting of polypeptides differing in a single amino acidfrom the normal polypeptide. The T cells recognising these polypeptidesin the context of MHC molecules on the surface of the tumour cells arecapable of killing the tumour cells and thus rejecting the tumour fromthe host (Boon et al., 1989, Cell 58: 293-303).

[0006] MHC molecules in humans are normally referred to as HLA (humanleukocyte antigen) molecules. There are two principal classes of HLAmolecules: class I and class II. HLA class I molecules are encoded byHLA A, B and C subloci and primarily activate CD8+ cytotoxic T cells.HLA class II molecules, on the other hand, primarily activate CD4+(cytotoxic or helper) T cells, and are encoded by the HLA DR, DP and DQsubloci. Every individual normally has six different HLA class Imolecules, usually two alleles from each of the three subgroups A, B andC, although in some cases the number of different HLA class I moleculesis reduced due to the occurrence of the same HLA allele twice. For ageneral review, see Roitt, I. M. et al. (1998) Immunology, 5^(th)Edition, Mosby, London.

[0007] The HLA gene products are highly polymorphic. Differentindividuals express distinct HLA molecules that differ from those foundin other individuals. This explains the difficulty of finding HLAmatched organ donors in transplantations. The significance of thegenetic variation of the HLA molecules in immunobiology lies in theirrole as immune-response genes. Through their polypeptide bindingcapacity, the presence or absence of certain HLA molecules governs thecapacity of an individual to respond to specific polypeptide epitopes.As a consequence, HLA molecules influence resistance or susceptibilityto disease.

[0008] T cells may inhibit the development and growth of cancer by avariety of mechanisms. Cytotoxic T cells, both HLA class I restrictedCD8+ and HLA class II restricted CD4+, may directly kill tumour cellspresenting the appropriate tumour antigens. Normally, CD4+ helper Tcells are needed for cytotoxic CD8+ T cell responses, but if thepolypeptide antigen is presented by an appropriate APC, cytotoxic CD8+ Tcells can be activated directly, which results in a quicker, strongerand more efficient response.

[0009] In International Application PCT/N092/00032 (published asWO92/14756), synthetic polypeptides and fragments of oncogene proteinproducts which have a point of mutation or translocations as compared totheir proto-oncogene or tumour suppressor gene protein are described.These polypeptides correspond to, completely cover or are fragments ofthe processed oncogene protein fragment or tumour suppressor genefragment as presented by cancer cells or other antigen presenting cells,and are presented as a HLA-polypeptide complex by at least one allele inevery individual. The polypeptides were shown to induce specific T cellresponses to the actual oncogene protein fragment produced by the cellby processing and presented in the HLA molecule. In particular, it isdescribed in WO92/14756 that polypeptides derived from the p21-rasprotein which had point mutations at particular amino acid positions,namely positions 12, 13 and 61. These polypeptides have been shown to beeffective in regulating the growth of cancer cells in vitro.Furthermore, the polypeptides were shown to elicit CD4+ T cell immunityagainst cancer cells harbouring the mutated p21-ras oncogene proteinthrough the administration of such polypeptides in vaccination or cancertherapy schemes. It has subsequently been shown that these polypeptidesalso elicit CD8+ T cell immunity against cancer cells harbouring themutated p21 ras oncogene protein through the administration mentionedabove (Gjertsen, M. K. et aL, 1997, Int. J Cancer 72: 784-790).

[0010] International Application PCT/NO99/00143 (published asWO99/58552) describes synthetic polypeptides and fragments of mutantprotein products arising from frameshift mutations occurring in genes incancer cells. These polypeptides correspond to, completely cover or arefragments of the processed frameshift mutant protein fragment aspresented by cancer cells or other antigen presenting cells, and arepresented as a HLA-polypeptide complex by at least one allele in everyindividual. In particular polypeptides resulting from frameshiftmutations in the BAX and hTGF□-RII genes are disclosed. Thesepolypeptides were shown to be effective in stimulating CD4+ and CD8+ Tcells in a specific manner.

[0011] However, the polypeptides described above will be useful only incertain numbers of cancers involving oncogenes with point mutations,frameshift mutations or translocation in a proto-oncogene or tumoursuppressor gene. There is a strong need for an anticancer treatment orvaccine that will be effective against a generic range of cancers.

[0012] The concerted action of a combination of altered oncogenes andtumour-suppressor genes results in cellular transformation anddevelopment of a malignant phenotype. Such cells are however prone tosenescence and have a limited life-span. In most cancers,immortalisation of the tumour cells requires the turning on of an enzymecomplex called telomerase. In somatic cells, the catalytic subunit ofthe telomerase holoenzyme, hTERT (human telomerase reversetranscriptase), is not normally expressed. Additional events, such asthe action of proteins encoded by a tumour virus or demethylation ofsilenced (methylated) promoter sites, can result in expression of thegenes encoding the components of the functional telomerase complex intumour cells.

[0013] Due to the presence of telomerase in most types of cancer cells,the enzyme has been disclosed as a general cancer vaccine candidate(International Patent Application No. PCT/NO99/00220, published asWO00/02581). WO00/02581 describes a method for preventing or treatingcancer by generating a T cell response against telomerase-expressingcells in a mammal suffering (or likely to suffer from) cancer. It isdemonstrated in WO00/02581 that both CD4+ and CD8+ T cells can bestimulated by administration of polypeptides having sequences derivedfrom such a telomerase protein.

[0014] Alternative splice variants of the telomerase pre-mRNA have beenreported in the literature (Kilian, A. et al., 1997, Hum. Mol. Genet. 6:2011-2019). Kilian et al. (1997, supra) indicated that it was noteworthythat several splice variants were located with the critical RT (reversetranscriptase) domain of hTERT. They stated, however, that a fullunderstanding of the significance of the hTERT splice variants was notobtained and that further functional characterisation was required.

[0015] Analysis of the complete genomic sequence of the hTERT gene, hasverified that the different mRNA splice variants arise from the usage ofalternative splice sites in the hTERT pre-mRNA (Wick, M. et al., 1999,Gene 232: 97-106). Compared with the full-length hTERT mRNA, at leastfive additional splice variants have been detected. A schematic drawingof these variants are provided in FIG. 1, and FIG. 2 shows an alignmentof the proteins encoded. Two of the splice variants, named α-del (orDEL1) and β-del (or DEL2), represent deletions of specific codingsequences. The α-del variant has deleted the first 36 nucleotides ofexon 6 and encodes a protein which lacks a stretch of 12 internal aminoacids. In the β-del variant 182 nucleotides representing the entireexons 7 and 8 are missing, leading to a shift in the open reading frameand a truncated protein with a 44-amino acid long carboxyl terminus notpresent in the full-length hTERT protein. The remaining splice variantsresult from the use of alternative splice sites located inside intronregions, resulting in the insertion of intron sequences within the openreading frame and premature termination of translation. The σ-insert (orINS1) variant results from an insertion of the first 38 nucleotides ofintron 4. The σ-insert does not contain a stop codon, but instead, theopen reading frame extends 22 nucleotides into the normal sequence usingan alternative reading frame. The γ-insert (or INS3) variant is causedby insertion of the last 159 nucleotides from intron 14. Ins-4 containsthe first 600 nucleotides from intron 14 while at the same time havingdeleted exon 15 and most of exon 16. The truncated proteins resultingfrom translation of these splice variants are shown in FIG. 2.

[0016] Several recent studies have addressed the regulation oftelomerase activity, and some correlation between hTERT mRNAtranscription and telomerase activity has been reported for several celllines and tissues (Nakamura, T. M. et al., 1997, Science 277: 955-959;Meyerson, M. et al., 1997, Int. J. Cancer 85: 330-335; Nakayama, J. etal., 1998, Nature Genet. 18: 65-68; Liu, K. et al., 1999, Proc. NatlAcad. Sci. USA 96: 5147-5152). Others studies have shown that telomeraseactivity is up-regulated through phosphorylation of the hTERT protein byprotein kinase Cα, and conversely, down-regulated by the presence ofprotein kinase C inhibitors and phosphatase 2A (Li, H. et al., 1997, J.Biol. Chem. 272: 16729-16732; Li, H. et al., 1998, J. Biol. Chem. 273:33436-33442; Bodnar, A. G. et al., 1996, Exp. Cell Res. 228: 58-64; Ku,W. C. et al., 1997, Biochem. Biophys. Res. Comm. 241: 730-736).Alternative splicing of the hTERT pre-mRNA represents an additionalmechanism for regulating telomerase activity, and has been shown tomediate down-regulation during fetal kidney development and in adultovarian and uterine tissues (Ulaner, G. A. et al., 1998, Cancer Res. 58:4168-4172; Ulaner, G. A. et al., 2000, Int. J. Cancer 85: 330-335). Thefocus of the abovementioned studies has been on the α and β splicevariants, presumably because they delete sequences which are believed toencode critical reverse transcriptase motifs (Lingner, J. et al., 1997,Science 276: 561-567).

[0017] The present invention provides peptides and nucleic acidsencoding said peptides based on the TERT γ and σ splice variants, andthe novel use of these peptides and nucleic acids in medicine.

[0018] Thus according to the present invention there is provided apolypeptide for use in medicine; wherein the polypeptide:

[0019] a) comprises a sequence given in SEQ ID NO: 1, 2, 3, 4, 5, 6 or11;

[0020] b) comprises 8 contiguous amino acids from SEQ ID NO: 1, 2, 3, 4,5, 6 or 11, with the proviso that at least one of said 8 contiguousamino acids is from SEQ ID NO: 1, 3, 5 or 11; or

[0021] c) comprises 8 contiguous amino acids that have only one, two orthree amino acid changes (eg. substitutions) relative to the 8contiguous amino acids as described in b) above, with the proviso thatthat at least one of the 8 contiguous amino acids present is from SEQ IDNO: 1, 3, 5 or 11;

[0022] wherein the polypeptide is capable of inducing a T cell response.

[0023] The term “comprises” used herein includes “consists”. Thepolypeptide (or nucleic acid) of the present invention may be flanked byone or more amino acid (or nucleic acid) residues unless otherwisespecified. For example, the polypeptide may be part of a fusion proteinwhich has one or more flanking domain at the N- or C-terminus to allowfor purification of the fusion protein.

[0024] Amino acid changes or modifications (eg. substitutions) in thepolypeptide may in particular be made to the anchor residues which fitinto HLA or MHC molecules for presentation to T cells. Enhanced bindingand immunogenic properties of the polypeptide to HLA or MHC moleculesmay thus be achieved (see Bristol, J. A. et al., 1998, J. Immunol.160(5): 2433-2441; Clay, T. M. et al., 1999, J. Immunol. 162(3):1749-1755).

[0025] The polypeptide described above optionally may:

[0026] a) have at least 55% sequence identity with a molecule comprisingthe sequence of SEQ ID NO: 1, as determined by an NCBI BLASTP Version2.1.2 search with default parameters;

[0027] b) have at least 55% sequence identity with a molecule comprisingthe sequence of SEQ ID NO: 2, as determined by an NCBI BLASTP Version2.1.2 search with default parameters;

[0028] c) have at least 40% sequence identity with a molecule comprisingthe sequence of SEQ ID NO: 3, as determined by an NCBI BLASTP Version2.1.2 search with an Expect value of 1000 and other parameters asdefault;

[0029] d) have at least 40% sequence identity with a molecule comprisingthe sequence of SEQ ID NO: 4, as determined by an NCBI BLASTP Version2.1.2 search with an Expect value of 1000 and other parameters asdefault;

[0030] e) have at least 70% sequence identity with a molecule comprisingthe sequence of SEQ ID NO: 5, as determined by an NCBI BLASTP Version2.1.2 search with an Expect value of 100000 and other parameters asdefault;

[0031] f) have at least 50% sequence identity with a molecule comprisingthe sequence of SEQ ID NO: 6, as determined by an NCBI BLASTP Version2.1.2 search with an Expect value of 10000 and other parameters asdefault; or

[0032] g) have at least 40% and preferably 60% sequence identity with amolecule comprising the sequence of SEQ ID NO: 11, as determined by anNCBI BLASTP Version 2.1.2 search with an Expect value of 1000 and otherparameters as default;

[0033] The NCBI BLASTP program can be found athttp://www.ncbi.nlm.nih.pov/blast/, and default parameters changed usingthe Advanced Search. Higher than default “Expect” values may be requiredwhen searching with small query sequences for matches to be displayed.The term “sequence identity” used herein refers to amino acid residuesin optimally aligned sequences which match exactly at correspondingrelative positions. For example, the NCBI BLASTP program provides apercentage value of identities between query and subject (“hit”)sequences.

[0034] The polypeptide described above may comprise a sequence as givenin SEQ ID NO: 1, 2, 3, 4, 5, 6 or 11 or may be a fragment of a sequenceas shown in SEQ ID NO: 1, 3, 5, 6 or 11.

[0035] While the polypeptides that are presented by HLA class IImolecules are of varying length (12-25 amino acids), the polypeptidespresented by HLA class I molecules must normally be nine amino acidresidues long in order to fit into the class I HLA binding groove. Alonger polypeptide will not bind if it cannot be processed internally byan APC or target cell, such as a cancer cell, before presenting in theclass I restricted HLA groove. Only a limited number of deviations fromthis requirement of nine amino acids have been reported, and in thosecases the length of the presented polypeptide has been either eight orten amino acid residues long. For reviews on polypeptide binding to MHCmolecules see Rammensee, H.-G. et al. (1995) Immunogenetics 41: 178-228and Barinaga (1992), Science 257: 880-881. Male, D. K. et al. (1996,Advanced Immunology, Mosby, London) provide background information onthe field of immunology.

[0036] The T cell response generated by the polypeptide described abovemay be generated after intracellular cleavage of the polypeptide toprovide a fragment that fits into an MHC or HLA binding groove.Alternatively, the polypeptide described above may not needintracellular cleavage to fit into an MHC or HLA class I binding groove.In this case, the polypeptide may be from 8 to 10 amino acids long. Alsoprovided is a polypeptide described above which does not needintracellular cleavage to fit into an MHC or HLA class II bindinggroove. In this case, the polypeptide may be from 12 to 25 amino acidslong.

[0037] The T cell response according to the present invention mayincrease the number and/or activity of T helper and/or T cytotoxiccells.

[0038] Also provided is a polypeptide which does not stimulate asubstantial cytotoxic T cell response in a patient against one or moreof the following: bone marrow stem cells, epithelial cells in coloniccrypts or lymphocytes.

[0039] Further provided according to the present invention is a nucleicacid molecule for use in medicine; wherein the nucleic acid molecule:

[0040] a) has a strand that encodes a polypeptide described above, asdescribed above;

[0041] b) has a strand that is complementary with a strand as describedin a) above; or

[0042] c) has a strand that hybridises with a molecule as described ina) or b) above (eg. under stringent conditions).

[0043] Stringent hybridisation conditions are discussed in detail at pp1.101-1.110 and 11.45-11.61 of Sambrook, J. et al. (1989, MolecularCloning, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor). One example of hybridisation conditions that can be usedinvolves using a pre-washing solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA(pH 8.0) and attempting hybridisation overnight at 55° C. using 5×SSC.Hybridising nucleic acid sequences within the scope of the presentinvention include probes, primers or DNA fragments. The term primerincludes a single stranded oligonucleotide which acts as a point ofinitiation of template-directed DNA synthesis under appropriateconditions (eg. in the presence of four different nucleosidetriphosphates and an agent for polymerisation, such as DNA or RNApolymerase or reverse transcriptase) in an appropriate buffer and at asuitable temperature.

[0044] Also provided is a vector or cell for use in medicine comprisinga nucleic acid molecule according to the present invention.

[0045] Further provided is a binding agent for use in medicine; whereinthe binding agent binds to a polypeptide described above as describedabove. Said binding agent may be specific for a polypeptide as describedabove. Said binding agent may be an antibody or a fragment thereof. Saidbinding agent may be lectin.

[0046] The term antibody in its various grammatical forms is used hereinto refer to immunoglobulin molecules and immunologically active portionsof immunoglobulin molecules, i.e., molecules that contain an antibodycombining site or paratope. Such molecules are also referred to as“antigen binding fragments” of immunoglobulin molecules. Illustrativeantibody molecules are intact immunoglobulin molecules, substantiallyintact immunoglobulin molecules and those portions of an immunoglobulinmolecule that contain the paratope, including those portions known inthe art as Fab, Fab′, F(ab′)2 and F(v). Antibodies of the presentinvention may be monoclonal or polyclonal. The term antibody is alsointended to encompass single chain antibodies, chimeric, humanised orprimatised (CDR-grafted) antibodies and the like, as well as chimeric orCDR-grafted single chain antibodies, comprising portions from twodifferent species. For preparation of antibodies see Harlow, E. andLane, D. (1988, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor) and Harlow, E. and Lane, D. (1999,Using Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor). Immunological adjuvants for vaccinescomprising lecithin may be used to stimulate antibody production (seefor example U.S. Pat. No. 4,803,070).

[0047] Further provided according to the present invention is a Tlymphocyte for use in medicine; wherein the T lymphocyte is capable ofkilling a cell expressing a polypeptide described above according to thepresent invention or of helping in the killing of such a cell. Said Tlymphocyte may be a T cytotoxic cell or a T helper cell.

[0048] Also provided is a clonal cell line for use in medicinecomprising a plurality of T lymphocytes as described above. Alsoprovided is a mixture of T lymphocytes for use in medicine comprising aT helper cell or a clonal cell line of such cells and a T cytotoxic cellor a clonal cell line of such cells.

[0049] Also provided is a method of generating T lymphocytes capable ofrecognising and destroying tumour cells in a mammal, comprising taking asample of T lymphocytes from a mammal and culturing the T lymphocytesample in the presence of at least one polypeptide described above in anamount sufficient to generate hTERT γ-insert protein specific Tlymphocytes and/or hTERT σ-insert protein specific T lymphocytes.

[0050] Also provided is a B lymphocyte which may be useful in generatingantibodies according to the present invention. Hybridomas which arecapable of generating antibodies according to the present invention arealso included (see for example Koehler et al., 1975, Nature 256:495-497; Kosbor et al., 1983, Immunol. Today 4: 72; Cote et al., 1983,PNAS USA 80: 2026-2030; Cole et al., 1985, Monoclonal Antibodies andCancer Therapy, Alan R. Liss Inc., New York, pp. 77-96).

[0051] Further provided according to the present invention is the use ofa polypeptide as described above, a nucleic acid as described above, avector or cell as described above, a binding agent as described above, aT lymphocyte as described above, a cell line as described above, or amixture of T lymphocytes as described above, in the preparation of amedicament for treating cancer, or in the preparation of a diagnosticfor diagnosing cancer. The cancer may be a mammalian cancer. Inparticular, the cancer may be human cancer. For example, the cancer maybe breast cancer, prostate cancer, pancreatic cancer, colo-rectalcancer, lung cancer, malignant melanoma, leukaemia, lymphoma, ovariancancer, cervical cancer or a biliary tract carcinoma.

[0052] Said medicament may be a vaccine.

[0053] The polypeptides described here are particularly suited for usein a vaccine capable of safely eliciting either CD4+ or CD8+ T cellimmunity. As the polypeptides may be synthetically produced, medicamentsincluding the polypeptides do not include transforming cancer genes orother sites or materials which might produce deleterious effects. Thepolypeptides may be targeted for a particular type of T cell responsewithout the side effects of other unwanted responses.

[0054] Said medicament may be an antisense molecule or is capable ofgenerating an antisense molecule in vivo.

[0055] Said diagnostic may be provided in a kit. The kit may comprisemeans for generating a detectable signal (eg. a fluorescent label, aradioactive label) or a detectable change (eg. an enzyme-catalysedchange). The kit may include instructions for use in diagnosing cancer.

[0056] Further provided is a pharmaceutical composition comprising apolypeptide as described above, a nucleic acid as described above, avector or cell as described above, a binding agent as described above, aT lymphocyte as described above, a cell line as described above, or amixture of T lymphocytes as described above.

[0057] Said pharmaceutical composition may comprise a polypeptidecapable of inducing a T cell response directed against a polypeptideproduced by an oncogene or against a mutant tumour suppressor protein,or a nucleic acid encoding such a polypeptide, or a binding agent thatbinds such a polypeptide, or a T cell that is capable of killing a cellexpressing such a polypeptide or of helping in the killing of such acell. Example of such oncogenes or mutant tumour suppressor proteinsinclude p21-ras, Rb, p53, abl, gip, gsp, ret or trk. The oncogene targetmay be the p21-ras polypeptides described in International ApplicationNo. PCT/NO92/00032 (Publication No. WO92/14756).

[0058] Also provided is a combined preparation comprising a componentfrom the pharmaceutical compositions described above for simultaneous,separate or sequential use in anticancer therapy.

[0059] Also provided is a pharmaceutical composition or a combinedpreparation as described above further comprising a pharmaceuticallyacceptable carrier, diluent, additive, stabiliser, and/or adjuvant; saidcomposition or combined preparation optionally further including one ormore of: a cytokine or growth factor (eg. IL-2, IL-12; and/or GM-CSF)and another polypeptide arising from a frameshift mutation (eg. aframeshift mutation in the BAX or hTGFβ-RII gene.)

[0060] The stimulatory effect on CD4+ and CD8+ T cells in a specificmanner by polypeptides resulting from frameshift mutations in the BAXand hTGFβ-RII genes was disclosed in WO99/58552 (see above).

[0061] The pharmaceutical composition or combined preparation describedabove may be a vaccine.

[0062] The pharmaceutical composition or combined preparation describedabove may comprise or be capable of producing antisense molecules.

[0063] Also provided is a method for the preparation of a pharmaceuticalcomposition as described above, comprising the steps of combining theabove described components with a pharmaceutically acceptable carrier,diluent, additive, stabiliser and/or adjuvant.

[0064] A pharmaceutical composition according to the present maycomprise any of the following mixtures:

[0065] a) a mixture of at least one polypeptide described above togetherwith another polypeptide having a different sequence;

[0066] b) a mixture of at least one polypeptide described above togetherwith another polypeptide having an overlapping sequence, so that thepolypeptides are suitable to fit different MHC or HLA alleles;

[0067] c) a mixture of both mixtures a) and b);

[0068] d) a mixture or several mixtures a);

[0069] e) a mixture of several mixtures b); or

[0070] f) a mixture of several mixtures a) and several mixtures b).

[0071] The polypeptides in the mixture may be covalently linked witheach other to form larger polypeptides or even cyclic polypeptides. Thepolypeptides themselves may be in a linear or cyclic form.

[0072] Also provided according to the present invention is a diagnosticcomposition comprising a polypeptide as described above, a nucleic acidas described above, a vector or cell as described above, a binding agentas described above, a T lymphocyte as described above, a cell line asdescribed above, or a mixture of T lymphocytes above.

[0073] Also provided according to the present invention is a diagnostickit as described above.

[0074] Also provided according to the present invention is a method oftreatment or prophylaxis of cancer of the human or animal bodycomprising administering a therapeutically effective amount ofpharmaceutical composition described above to a patient or animal inneed of same. The invention includes a method of treatment orprophylaxis of patient or animal afflicted with cancer, the methodcomprising administering to said patient or animal a pharmaceuticalcomposition described above in an amount sufficient to elicit a T-cellresponse against said cancer. The method of treatment may also includestimulation in vivo or ex vivo with a pharmaceutical compositiondescribed above. Ex vivo therapy may include isolating dendritic cellsor other suitable antigen presenting cells from a patient or animal,loading said cells with at least one polypeptide or nucleic aciddescribed above, and infusing these loaded cells back into the patientor animal. The polypeptides or nucleic acids described above may also beused in a method of vaccination of a patient in order to obtainresistance against cancer. Oncogenes are sometimes associated withviruses. The present invention is also suitable for the treatment ofcertain viral disorders.

[0075] The polypeptides according to the present invention may beadministered in an amount in the range of 1 microgram (1 μg) to 1-gram(1 g) to an average human patient or individual to be vaccinated. It ispreferred to use a smaller dose in the range of 1 microgram (1 μg) to 1milligram (1 mg) for each administration.

[0076] The exact dosages, ie. pharmaceutically acceptable dosages, andadministration regime of pharmaceutical compositions and medicaments ofthe present invention may be readily determined by one skilled in theart, for example by using for example dose-response assays.

[0077] The administration may take place one or several times assuitable to establish and/or maintain the desired T cell immunity. Thepolypeptides according to the present invention may be administeredtogether, either simultaneously or separately, with compounds such ascytokines and/or growth factors, i.e., interleukin-2 (IL-2),interleukin-12 (IL-12), granulocyte macrophage colony stimulating factor(GM-CSF) or the like in order to strengthen the immune response as knownin the art. The polypeptides can be used in a vaccine or a therapeuticcomposition either alone or in combination with other materials. Forexample, the polypeptide or polypeptides may be supplied in the form ofa lipopeptide conjugate which is known to induce a high-affinitycytotoxic T cell response (Deres, K. et al., 1989, Nature 342: 561-564).

[0078] The polypeptides according to the present invention may beadministered to an individual or animal in the form of DNA vaccines. TheDNA encoding the polypeptide(s) may be in the form of cloned plasmid DNAor synthetic oligonucleotide. The DNA may be delivered together withcytokines, such as IL-2, and/or other co-stimulatory molecules. Thecytokines and/or co-stimulatory molecules may themselves be delivered inthe form of plasmid or oligonucleotide DNA.

[0079] Response to a DNA vaccine has been shown to be increased by thepresence of immunostimulatory DNA sequences (ISS). These can take theform of hexameric motifs containing methylated CpG, according to theformula: 5′-purine-purine-CG-pyrimidine-pyrimidine-3′. DNA vaccinesaccording to the present invention may therefore incorporate these orother ISS, in the DNA encoding the hTERT γ-insert protein and/or thehTERT σ-insert protein, in the DNA encoding the cytokine or otherco-stimulatory molecules, or in both. A review of the advantages of DNAvaccination is provided by Tighe et al. (1998, Immunology Today, 19(2):89-97).

[0080] Also provided according to the present invention is thepolypeptide as described above, optionally in isolated form, wherein thepolypeptide is not a polypeptide consisting of the sequences shown inFIG. 4.

[0081] The polypeptide sequence shown in FIG. 4 represents thedisclosure in FIG. 5C of Kilian et al. (1997, supra) of 46 amino acidresidues at the C-terminal end of the circa 1100 amino acid residuehTERT γ-insert splice variant, which includes 44 amino acids of SEQ IDNO: 1. The sequence provided in Kilian et al. (1997, supra) shows the“alternative C-terminus” of the hTERT γ-insert splice variant protein.(Kilian et al., 1997, supra, indicate that the corresponding DNAsequence in provided by GenBank Accession number AF015950.) Kilian etal. (1997, supra) do not disclose as a separate entity the polypeptideaccording to SEQ ID NOs: 1, 2 or 5 at the C-terminal end of the hTERTγ-insert splice variant protein, and they do not disclose or suggestmedicinal use of the polypeptide according to SEQ ID NO: 1, 2 or 5.

[0082] The polypeptides described herein may be produced by conventionalprocesses, for example, by the various polypeptide synthesis methodsknown in the art. Alternatively, they may be fragments of a hTERTγ-insert protein and/or a hTERT σ-insert protein produced by cleavage,for example, using cyanogen bromide, and subsequent purification.Enzymatic cleavage may also be used. The hTERT γ-insert protein and thehTERT σ-insert protein or peptides may also be in the form ofrecombinant expressed proteins or polypeptides.

[0083] Also provided is the nucleic acid as described herein, optionallyin isolated form; wherein the nucleic acid is not a nucleic acidencoding the polypeptide excluded above and is also not a nucleic acidas shown in FIG. 4 or FIG. 5.

[0084] The nucleic acid sequence at the 3′-end of the circa 3100 bphTERT γ-insert splice variant, part of which encodes the C-terminal endof the corresponding protein that includes SEQ ID NO: 1, is provided inFIG. 4 of Kilian et al. (1997, supra). The nucleic acid sequence at theexon-intron borders of the hTERT splice variants INS1 (equivalent to theσ-insert splice variant) and INS3 (equivalent to the γ-insert splicevariant), as disclosed in FIG. 2B of Wick et al. (1999, supra), areshown in FIG. 5. The nucleic acids shown in FIG. 5 as disclosed in FIG.2B of Wick et al. (1999, supra) include nucleotides which encode aminoacid residues present in SEQ ID NOs: 1-6 and 11. Wick et al. (1999,supra) make no specific reference to the existence of the nucleic acidsshown as distinct entities or to their medical use. Wick et al., 1999,supra, provide reference to the complete nucleotide sequence of theirhTERT gene in GenBank Accession numbers AF128893 and AF128894.

[0085] Nucleic acids encoding the polypeptides of the present inventionmay be made by oligonucleotide synthesis. This may be done by any of thevarious methods available in the art. A nucleic acid encoding telomeraseprotein may be cloned from a genomic or cDNA library, using conventionallibrary screening. The probe may correspond to a portion of any sequenceof a known hTERT γ-insert and/or hTERT σ-insert gene. Alternatively, thenucleic acid can be obtained by using the Polymerase Chain Reaction(PCR). The nucleic acid is preferably DNA, and may suitably be clonedinto a vector. Subclones may be generated by using suitable restrictionenzymes. The cloned or subcloned DNA may be propagated in a suitablehost, for example a bacterial host. Alternatively, the host can be aeukaryotic organism, such as yeast or baculovirus. The hTERT γ-insertand the hTERT σ-insert proteins or polypeptides may be produced byexpression in a suitable host. In this case, the DNA is cloned into anexpression vector. A variety of commercial expression kits areavailable. The methods described in Sambrook, J. et al. (1989, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor) may be used for these purposes.

[0086] Also provided is the vector or cell as described herein,optionally in isolated form.

[0087] Further provided is the binding agent as described herein,optionally in isolated form.

[0088] Yet further provided is the T lymphocyte as described herein,optionally in isolated form.

[0089] Also provided is the clonal cell line as described herein,optionally in isolated form.

[0090] Further provided is the mixture of T lymphocytes as describedherein.

[0091] Also provided is a machine readable data carrier (eg. a disk)comprising the sequence of a polypeptide or of a nucleic acid asdescribed herein.

[0092] Yet further provided is a method comprising using the sequence ofa polypeptide or a nucleic acid molecule as described herein to performsequence identity studies, sequence homology studies, or hybridisationstudies. Said method may include using said sequence to predictstructure and/or function (eg. to predict anti-cancer activity). Alsoprovided is the use of this method in a drug development or screeningprocedure. Further provided is a drug identified or selected by thisprocedure.

[0093] Also provided is a computer or database that displays or stores asequence of a polypeptide or a nucleic acid molecule as described hereinor that is set up to perform a method as described above.

[0094] Also provided is the invention as substantially hereinbeforedescribed with reference to the accompanying figures and examples.

[0095] The phrases “amino acid residue” and “amino acid” are broadlydefined to include modified and unusual amino acids as defined in WIPOStandard ST.25, and incorporated herein by reference.

[0096] The term treatment or therapy used herein includes prophylactictreatment or therapy where applicable.

[0097] The contents of each of the references discussed herein,including the references cited therein, are herein incorporated byreference in their entirety.

[0098] The invention will be further apparent from the followingdescription, with reference to the several accompanying figures, whichshow, by way of example only, various polypeptides and their useaccording to the present invention.

[0099] Of the figures:

[0100]FIG. 1 is a schematic drawing of the full-length hTERT mRNA andsplice variants found in cancer cell lines;

[0101]FIG. 2 shows a protein alignment between a portion of the hTERTprotein and proteins resulting from translation of splice variants;

[0102]FIG. 3 shows the carboxyl termini of the hTERT γ-insert andσ-insert splice variant proteins;

[0103]FIG. 4 shows prior art sequences relating to the hTERT γ-insertsplice variant as disclosed in FIG. 5C of Kilian et al. (1997, supra);

[0104]FIG. 5 show prior art sequences relating to the hTERT γ-insert andσ-insert splice variants as disclosed in FIG. 2B of Wick et al. (1999,supra);

[0105]FIG. 6 shows results from RT-PCR analysis of the regionscomprising the γ-insert (A) and σ-insert (B) splice variants of hTERT;

[0106]FIG. 7 shows proliferative T cell responses induced in human bloodsamples by a polypeptide having the amino acid sequence of SEQ ID NO:11;

[0107]FIG. 8 shows proliferation of T cell clones induced by apolypeptide having the amino acid sequence of SEQ ID NO:11; and

[0108]FIG. 9 shows proliferation of other T cell clones induced by apolypeptide having the amino acid sequence of SEQ ID NO:11.

[0109] In FIG. 1, the position of introns present in the hTERT pre-mRNAis indicated by the letter “i” followed by an appropriate number.Insertion and deletion variants are shown as square boxes; shaded fillrepresents sequences that encode protein sequence not present in thefull-length hTERT protein. Position and orientation of oligonucleotideprimers used to analyse the different splice variants is indicated byarrows.

[0110] In FIG. 2, amino acid numbering is shown above the sequence.Amino acids are represented by their standard one letter abbreviationknown in the art.

[0111] In FIG. 3, SEQ ID NO: 1 reflects the truncated tail of the hTERTγ-insert protein and SEQ ID NO:2 reflects the same polypeptide with anextension at the amino terminus with the nine amino acids normally foundin these positions in the naturally occurring hTERT γ-insert expressionproduct (underlined). SEQ ID NO: 3 reflects the truncated tail of thehTERT σ-insert protein. SEQ ID NO: 4 reflects SEQ ID NO: 3 with anextension at the amino terminus with the nine amino acids normally foundin these positions in the naturally occurring hTERT σ-insert expressionproduct (underlined).

[0112] In FIG. 4, the exon/intron junctions of insert splice variant 3(equivalent to the hTERT γ-insert splice variant) is shown as providedin FIG. 5C of Kilian et al. (1997, supra). The following information isprovided by Kilian et al. (1997, supra): the nucleic acid sequence isshown above a protein translation sequence, with the putative unsplicedintron given in bold type; putative exon/intron junctions are markedwith |; the nucleic acid sequence numbering corresponds as follows:nucleotide 1 corresponds to nucleotide 139 of the sequence in GenBankAccession number AF015950; and amino acids corresponding to the putativec-Abl/SH3 binding site are underlined. The amino acid sequence shown byKilian et al. (1997; supra) represents the C-terminal end of a circa1100 amino acid residue hTERT γ-insert splice variant protein.

[0113] In FIG. 5, nucleotides of the exon-intron borders of the hTERTsplice variants INS1 equivalent to the σ-insert splice variant) and INS3(equivalent to the γ-insert splice variant), as disclosed in FIG. 2B ofWick et al. (1999, supra), are represented. Intronic and exonicsequences are shown in lower-case and upper-case letters, respectively.Wick et al. (1999, supra) indicate that the nucleotide sequence of theirhTERT gene has been deposited as GenBank Accession numbers AF128893 andAF128894.

[0114] It has been established that the hTERT γ-insert and σ-insertsplice variants are expressed in cancer cell lines and tumours but areundetectable, or present at very low levels, in normal cells. Thepresent application therefore discloses general cancer vaccinecandidates with improved specificity in comparison with vaccines basedon the functional variant of the telomerase (hTERT) protein. Both γ andσ inserts results in formation of an early stop codon and the expressionof a protein that is truncated at the carboxyl terminus. The truncatedhTERT γ- and σ-insert proteins have no telomerase activity themselves.In the case of a γ-insert the truncated tail of the protein is asequence of 44 amino acids (SEQ ID NO: 1). A σ-insert results in aprotein in which the truncated tail is a sequence of 20 amino acids (SEQID NO: 3). They are predominantly expressed in cancer cell lines but areundetectable, or present at very low levels, in normal cells and aretherefore targets for specific immunotherapy. According to the presentinvention, polypeptides corresponding to the carboxyl end (truncatedtail) of proteins expressed by hTERT γ-insert and/or σ-insert splicevariants, are useful as anticancer agents or vaccines with the functionto trigger the cellular arm of the immune system (T-cells) in humansagainst cancer cells. In a preferred embodiment of the invention, thepolypeptide comprises the sequence according to SEQ ID NO: 5. In anotherpreferred embodiment of the invention, the polypeptide comprises thesequence according to SEQ ID NO: 6. It yet another preferred embodiment,the polypeptide comprises the sequence according to SEQ ID NO: 11.

EXPERIMENTAL

[0115] The experiments outlined herein describe the characterisation ofhTERT splice variants in various cancer cell lines compared with normalcells. Synthesis of polypeptides according to the present invention, andexperiments for testing the efficacy of the polypeptides for use incancer therapy are detailed. An experiment showing induction andproliferation of human T cells by the peptide having an amino acidsequence according to SEQ ID NO: 11 is described.

[0116] RT-PCR Analysis of the γ-insert and σ-insert Splice Variants ofhTERT

[0117] RNA Analysis:

[0118] Poly(A)⁺ mRNA from completely lysed cells was isolated directlyfrom crude lysates using magnetic oligo(dT) beads (Dynal A S; Sakobsen,K. S. et al., 1990, Nucleic Acids Res. 18: 3669). Cytosolic mRNAfractions were prepared by incubating cells in 1% IGEPAL (Sigma) at 0°C. for one minute, followed by centrifugation [1000 g; 1 min.; 4° C.] toremove nuclei. Poly(A)⁺ mRNA was then isolated from the supernatantusing oligo(dT) beads as described above.

[0119] cDNA Synthesis and PCR:

[0120] First strand cDNA synthesis was carried out by standardprocedures using M-MLV RNaseH÷ reverse transcriptase (Promega Corp.),and the PCR reactions were performed by using HotStar Taq DNA polymerase(Qiagen) and run for 35 cycles on a PTC-200 thermal cycler (MJResearch). To obtain detectable products from PBM and CD34+ cells, 10%of the reaction was used as template in a second PCR reaction andamplified by 15 additional cycles.

[0121] For analysis of the γ-insert splice variant the plus-strandprimer variant the plus-strand primer hTERT-p3195 (5-GCC TCC CTC TGC TACTCC ATC CT—SEQ ID NO: 7) and minus-strand primer hTERT-m3652 (5-CGT CTAGAG CCG GAC ACT CAG CCT TCA—SEQ ID NO: 8) were used. Applied on thefull-length hTERT cDNA and the γ-insert variant, these primers producefragments of 465 and 624 nucleotides, respectively. The analysis of theσ-insert variant was performed by using primers hTERT-P6 (5-GCC AAG TTCCTG CAC TGG CTG A—SEQ ID NO: 9) and hTERT-m2044 (5-GCT CTA GAA CAG TGCCTT CAC CCT CG—SEQ ID NO: 10). The amplification product resulting fromusing these primers with full-length hTERT cDNA and the σ-insert variantcomprises 369 and 407 nucleotides, respectively. To verify that thesePCR products represent genuine splice variants, the fragments wereisolated from the gel and analysed by direct sequencing using an ABIprism 310 automated sequencer (PE Corp.).

[0122] Results:

[0123] Telomerase activity is subject to complex regulation at thepost-transcriptional level, and methods used to detect the presence orabsence of telomerase proteins should involve direct measurements of theprotein itself, or alternatively, mRNA variants. Furthermore, theabundance of the different hTERT splice variants found in cells is notnecessarily correlated with the levels found in the cytosolic fractionof the same cells (see FIG. 6). Such deviations may be explained bydifferences in the efficiency with which mRNA variants are transportedfrom the nucleus to the cytosolic compartment, and/or by differentialstability of the specific splice variants in the cytosol. It is wellknown in the art that such mechanisms are part of the concept of generegulation. Nevertheless, the studies conducted to explain hTERTregulation, including those cited above, have used total RNA or mRNAisolated from completely lysed cells for their analysis. Kits andreagents required to perform this kind of RNA isolation are widelyavailable in the commercial market. To obtain a correct picture of geneexpression, studies on mRNA abundance should include analysis of mRNAspecific to the cytosolic compartment.

[0124]FIG. 6 shows results from RT-PCR analysis of the regionscomprising the γ-(A) and σ-insert variants (B) of hTERT. HL60, K562, andJurkat denote the cancer cell lines analysed. HL60 is a promyelocyticleukemia cell line (Sokoloski, J. A. et al., 1993, Blood 82: 625-632),K562 an erythroid leukemia cell line (Lozzio, C. B. et al., 1975, Blood45: 321-324), while Jurkat is derived from acute T-lymphocyte leukemiacells (Gillis, S. et al., 1980, J. Exp. Med. 152: 1709-1719). The HL60,K562, and Jurkat cancer cell lines are commercially available (forexample, from ATCC, Oslo). PBM1, PBM2, PBM3 and PBM4 representperipheral blood mononuclear (PBM) cell populations isolated from fourdifferent healthy donors. CD34 denotes CD34-positive stem cells isolatedfrom a healthy donor, and CC1/CC2 denotes colon cancer biopsies obtainedfrom two cancer patients at the Norwegian Radium Hospital, Oslo, withCC2a and CC2b being two tissue samples dissected from the same tumour.RT-PCR reactions performed with mRNA isolated by complete lysis of cellsand with mRNA isolated from cytosolic fractions are marked with theletters “T” and “C”, respectively. “M” indicates lane with molecularweight marker. Position of PCR fragments representing the γ- andσ-insert splice variants and the respective full-length hTERT products(+) is indicated on the right side of the panels.

[0125] The RT-PCR analysis showed that both γ- and σ-insert splicevariants were readily detectable in all cancer cell lines and in one ofthe tumour samples analysed (CC2b), and with the σ-insert variantappearing as the most abundant in cytosolic fractions. In contrast, wewere not able to detect these variants in cytosolic mRNA populationsisolated from PBM cells despite the extensive PCR amplificationperformed with these samples. The identity of the weak 395-bp fragmentproduced with the σ-insert primers on PBM and CD34-positive cells is atpresent unknown.

[0126] Polypeptide Synthesis and Analysis for Applications Relating toCancer

[0127] Polypeptide Synthesis:

[0128] The polypeptides were synthesised by using continuous flow solidphase peptide synthesis. N-a-Fmoc-amino acids with appropriate sidechain protection were used. The Fmoc-amino acids were activated forcoupling as pentafluorophenyl esters or by using either TBTU ordiisopropyl carbodiimide activation prior to coupling. 20% piperidine inDMF was used for selective removal of Fmoc after each coupling. Cleavagefrom the resin and final removal of side chain protection was performedby 95% TFA containing appropriate scavengers. The polypeptides werepurified and analysed by reversed phase HPLC. The identity of thepolypeptides was confirmed by using electro-spray mass spectroscopy.

[0129] Polypeptide Testing and Cancer Therapy:

[0130] In order for a cancer vaccine according to the present invention,and methods for specific cancer therapy based on T cell immunity to beeffective, two conditions must be met:

[0131] (a) the polypeptide is at least 8 amino acids long and is afragment of the hTERT γ-insert protein or the hTERT σ-insert protein and

[0132] (b) the polypeptide is capable of inducing, either in its fulllength or after processing by antigen presenting cell, T cell responses.

[0133] The following experimental methods may be used to determine ifthese two conditions are met for a particular polypeptide. First, itshould be determined if the particular polypeptide gives rise to T cellimmune responses in vitro. It will also need to be established if thesynthetic polypeptides correspond to, or are capable after processing toyield, polypeptide fragments corresponding to polypeptide fragmentsoccurring in cancer cells harbouring the hTERT γ-insert protein and/orthe hTERT σ-insert protein or antigen presenting cells that haveprocessed naturally occurring hTERT γ-insert protein and/or hTERTσ-insert protein. The specificity of T cells induced in vivo by hTERTγ-insert and/or hTERT σ-insert polypeptide vaccination may also bedetermined.

[0134] In Vitro T Cell Response Analysis;

[0135] It is necessary to determine if hTERT γ-insert and/or hTERTσ-insert expressing tumour cell lines can be killed by T cell clonesobtained from peripheral blood from carcinoma patients after hTERTγ-insert and/or hTERT σ-insert polypeptide vaccination. T cell clonesare obtained after cloning of T-cell blasts present in peripheral bloodmononuclear cells (PBMC) from a carcinoma patient after hTERT γ-insertand/or hTERT σ-insert polypeptide vaccination. The polypeptidevaccination protocol includes several in vivo injections of polypeptidesintracutaneously with GM-CSF or another commonly used adjuvant. Cloningof T cells is performed by plating responding T cell blasts at 5 blastsper well onto Terasaki plates. Each well contains 2×10⁴ autologous,irradiated (30 Gy) PBMC as feeder cells. The cells are propagated withthe candidate hTERT γ-insert and/or hTERT σ-insert polypeptide at 25 μMand 5 U/ml recombinant interleukin-2 (rIL-2) (Amersham, Aylesbury, UK)in a total volume of 20 ml. After 9 days T cell clones are transferredonto flat-bottomed 96-well plates (Costar, Cambridge, Mass) with 1 mg/mlphytohemagglutinin (PHA, Wellcome, Dartford, UK), 5 U/ml rIL-2 andallogenic irradiated (30 Gy) PBMC (2×10⁵) per well as feeder cells.Growing clones are further expanded in 24-well plates with PHA/rIL-2 and1×10⁶ allogenic, irradiated PBMC as feeder cells and screened forpolypeptide specificity after 4 to 7 days.

[0136] T cell clones are selected for further characterisation. Thecell-surface phenotype of the T cell clone is determined to ascertain ifthe T cell clone is CD4+ or CD8+. T cell clone is incubated withautologous tumour cell targets at different effector to target ratios todetermine if lysis of tumour cells occurs. Lysis indicates that the Tcell has reactivity directed against a tumour derived antigen, forexample, hTERT γ-insert and/or hTERT σ-insert proteins.

[0137] Correlation between Polypeptides and in vivo hTERT InsertFragments;

[0138] In order to verify that the antigen recognised is associated withhTERT γ-insert protein or hTERT σ-insert protein, and to identify theHLA class I or class II molecule presenting the putative hTERT γ-insertor hTERT σ-insert polypeptide to the T cell clone, different hTERTγ-insert and/or hTERT σ-insert expressing tumour cell lines carrying oneor more HLA class I or II molecules in common with those of the patient,are used as target cells in cytotoxicity assays. Target cells arelabelled with ⁵¹Cr or ³H-thymidine (9.25×10⁴ Bq/mL) overnight, washedonce and plated at 5000 cells per well in 96 well plates. T cells areadded at different effector to target ratios and the plates areincubated for 4 hours at 37° C. and then harvested before counting in aliquid scintillation counter (Packard Topcount). For example, thebladder carcinoma cell line T24 (12Val⁺, HLA-A1⁺, B35⁺), the melanomacell line FMEX (12Val⁺, HLA-A2⁺, B35⁺) and the colon carcinoma cell lineSW 480 (12Val⁺, HLA-A2⁺, B8⁺) or any other telomerase positive tumourcell line may be used as target cells. A suitable cell line which doesnot express hTERT γ-insert and/or hTERT σ-insert proteins may be used asa control, and should not be lysed. Lysis of a particular cell lineindicates that the T cell clone being tested recognises anendogenously-processed hTERT γ-insert and/or hTERT σ-insert epitope inthe context of the HLA class I or class II subtype expressed by thatcell line.

[0139] Characterisation of T Cell Clones:

[0140] The HLA class I or class II restriction of a T cell clone may bedetermined by blocking experiments. Monoclonal antibodies against HLAclass I antigens, for example the panreactive HLA class I monoclonalantibody W6/32, or against class II antigens, for example, monoclonalsdirected against HLA class II DR, DQ and DP antigens (B8/11, SPV-L3 andB7/21), may be used. The T cell clone activity against the autologoustumour cell line is evaluated using monoclonal antibodies directedagainst HLA class I and class II molecules at a final concentration of10 μg/ml. Assays are set up as described above in triplicate in 96 wellplates and the target cells are preincubated for 30 minutes at 37° C.before addition of T cells.

[0141] The fine specificity of a T cell clone may be determined usingpolypeptide pulsing experiments. To identify the hTERT γ-insert and/orhTERT σ-insert polypeptide actually being recognised by a T cell clone,a panel of nonamer polypeptides is tested. ⁵¹Cr or ³H-thymidinelabelled, mild acid eluted autologous fibroblasts are plated at 2500cells per well in 96 well plates and pulsed with the polypeptides at aconcentration of 1 μM together with b2-microglobulin (2.5 μg/mL) in a 5%CO₂ incubator at 37° C. before addition of the T cells. Assays are setup in triplicate in 96 well plates and incubated for 4 hours with aneffector to target ratio of 5 to 1. Controls can include T cell clonecultured alone, with APC in the absence of polypeptides or with anirrelevant melanoma associated polypeptide MART-1/Melan-A polypeptide.

[0142] An alternative protocol to determine the fine specificity of a Tcell clone may also be used. In this alternative protocol, the TAPdeficient T2 cell line is used as antigen presenting cells. This cellline expresses only small amounts of HLA-A2 antigen, but increasedlevels of HLA class I antigens at the cell surface can be induced byaddition of b2-microglobulin. ³H-labelled target cells are incubatedwith the different test polypeptides and control polypeptides atconcentration of 1 μM together with b2-microglobulin (2.5 μg/mL) for onehour at 37° C. After polypeptide pulsing, the target cells are washedextensively, counted and plated at 2500 cells per well in 96 well platesbefore addition of the T cells. The plates are incubated for 4 hours at37° C. in 5% CO₂ before harvesting. Controls include T cell clonecultured alone or with target cells in the absence of polypeptides.Assays were set up in triplicate in 96 well plates with an effector totarget ratio of 20 to 1.

[0143] The sensitivity of a T cell clone to a particular polypeptideidentified above may also be determined using a dose-responseexperiment. Polypeptide sensitised fibroblasts can be used as targetcells. The target cells are pulsed with the particular peptide asdescribed above for fine specificity determination, with the exceptionthat the peptides are added at different concentrations before theaddition of T cells. Controls include target cells alone and targetcells pulsed with the irrelevant melanoma associated peptideMelan-A/Mart-1.

[0144] Induction and Proliferation of Human T Cell Response to the hTERTσ-insert Peptide

[0145] In this experiment, peripheral blood mononuclear cells (PBMC)from four healthy humans (donors “14328”, “14313”, “23244” and “23255”)and were isolated and primed for seven days with dendritic cells pulsedwith the SEQ ID NO: 11 peptide derived from the hTERT σ-insertpolypeptide, followed by two cycles consisting of seven daysre-stimulation with peptide-pulsed autologous PBMC. The dendritic cellswere derived from monocytes from peripheral blood. T cells from theresulting bulk culture were tested in triplicate with or withoutpeptide-pulsed antigen presenting cells (APC) before harvesting after 3days. To measure the proliferative capacity of the cultures,³H-thymidine (3.7×10⁴ Bq/well) was added to the culture overnight beforeharvesting. Cultures with non-pulsed APC or without APC served ascontrols. The results showing the proliferative capacity of the culturesare shown in FIG. 7. Further details of the protocol used are set outbelow.

[0146] T cell clones were obtained from the resulting bulk cultures fromnon-vaccinated donors 14313 and 23255. The clones were obtained from Tcell blasts preset in PRMCs as described in the above section “In vitroT cell response analysis”. The results of proliferation of the T cellclones with peptide-pulsed and non-peptide pulsed anitgen presentingcells are shown in FIG. 8 (donor 14313) and FIG. 9 (donor 23255).

[0147] Results in FIGS. 7, 8 and 9 are given as mean counts per minute(cpm) of triplicate measurements. The data demonstrates that blood fromhumans contain circulating T cells specific for a peptide (SEQ ID NO:11) derived from the peptide derived from the hTERT σ-insertpolypeptide, and furthermore that such T cells can be expanded in vitrofollowing stimulation with the relevant peptide.

[0148] Thus, the experiments of FIGS. 7, 8 and 9 show that the hTERTσ-insert polypeptide is immunogenic in man. In vitro (or in vivo)stimulation can this give rise to hTERT σ-insert protein-specific T cellresponses with the potential to recognise the same antigen whenoverexpressed by a tumour growing in a cancer patient. This particularexperiment demonstrates that in principle the peptide of SEQ ID NO: 11could be developed as a cancer vaccine in humans.

[0149] Protocol for Induction of MHC Class II Restricted T Cell Response

[0150] Day 0:

[0151] PBMCs were separated out from 50 ml of blood (from buffy coat).The cells were counted and re-suspended in complete RPMI-1640/15% poolserum.

[0152] Bulk cultures were set up with 1-2 wells on a 24-well plate ofPBMCs at 2×10⁶ cells/ml in 1-1.5 ml. 25 μM of SEQ ID NO:11 peptidederived from the hTERT σ-insert polypeptide were added.

[0153] Day 9-10:

[0154] Bulk cultures were harvested and stimulated with irradiated PBMCsand peptide. If there was high cell death, cultures were lymphoprepseparated, otherwise they were counted and resuspended in RPMI/15% poolserum. (Lymphoprep centrifugation of bulk cultures is carried out in 15ml Falcon tubes by 1; adding 8 ml of cell suspension, and 2; underlaywith 2 ml of lymphoprep. Spin at 1500 rpm for 30 min, and wash twicewith salt water.) One vial of autologous PBMCs was defrosted, washed,counted and resuspended in RPMI/15% FCS. The PBMCs were irradiated (25GY, 5 min 58 sec). Cells were plated out in 24-well plates. 0.5-2.0×10⁶T cells from bulk cultures were stimulated with 1×10⁶ irradiated feedercells (PBMCs) and 25 μM SEQ ID NO:11 peptide. The final volume was 1 ml.

[0155] Day 12:

[0156] IL-2 (10 U/ml) was added. Medium was also added if necessary byreplacing half the volume. Cultures were split if necessary.

[0157] Day 17:

[0158] The T cells in bulk culture were re-stimulated as on day 10, withautologous, irradiated PBMC's and SEQ ID NO: 11 peptide.

[0159] Day 19:

[0160] IL-2 (10 U/ml) was added to day 17 re-stimulated bulk cultures.

[0161] Day 24:

[0162] A proliferation assay for testing T cells for peptide specificitywas set up in 96-well plates, each condition in triplicate: Triplicates1-3 4-6 7-9 10-12 Controls PBMC¹ Tc² PBMC¹ + PBMC¹ + Tc² Tc² + IL-2³Test PBMC¹ + PBMC¹ + Tc² + samples Tc² + Pep⁴ Pep⁴ + IL-2³

[0163] On day 2-3 of proliferation assay, ³H-Thymidine (20 μl) wasadded, and incubated at 37 C overnight before harvesting.

[0164] In a variant of the protocol (as used in the above example) thePBMCs were, on Day 0, primed with dendritic cells pulsed with SEQ ID NO:11.

1 11 1 44 PRT Homo sapiens 1 Ala Glu Glu Asn Ile Ser Val Val Thr Pro AlaVal Leu Gly Ser Gly 1 5 10 15 Gln Pro Glu Met Glu Pro Pro Arg Arg ProSer Gly Val Gly Ser Phe 20 25 30 Pro Val Ser Pro Gly Arg Gly Ala Gly LeuGly Leu 35 40 2 53 PRT Homo sapiens 2 Tyr Ser Ile Leu Lys Ala Lys AsnAla Ala Glu Glu Asn Ile Ser Val 1 5 10 15 Val Thr Pro Ala Val Leu GlySer Gly Gln Pro Glu Met Glu Pro Pro 20 25 30 Arg Arg Pro Ser Gly Val GlySer Phe Pro Val Ser Pro Gly Arg Gly 35 40 45 Ala Gly Leu Gly Leu 50 3 20PRT Homo sapiens 3 Val Ala Val Leu Trp Phe Asn Phe Leu Phe Lys Gln LysPro Ser Val 1 5 10 15 Ser Pro Arg Gly 20 4 29 PRT Homo sapiens 4 Ala ArgThr Phe Arg Arg Glu Lys Arg Val Ala Val Leu Trp Phe Asn 1 5 10 15 PheLeu Phe Lys Gln Lys Pro Ser Val Ser Pro Arg Gly 20 25 5 9 PRT Homosapiens 5 Phe Leu Phe Lys Gln Lys Pro Ser Val 1 5 6 18 PRT Homo sapiens6 Arg Val Ala Val Leu Trp Phe Asn Phe Leu Phe Lys Gln Lys Pro Ser 1 5 1015 Val Ser 7 23 DNA Homo sapiens 7 gcctccctct gctactccat cct 23 8 27 DNAHomo sapiens 8 cgtctagagc cggacactca gccttca 27 9 22 DNA Homo sapiens 9gccaagttcc tgcactggct ga 22 10 26 DNA Homo sapiens 10 gctctagaacagtgccttca ccctcg 26 11 21 PRT Homo sapiens 11 Arg Val Ala Val Leu TrpPhe Asn Phe Leu Phe Lys Gln Lys Pro Ser 1 5 10 15 Val Ser Pro Arg Gly 20

1. A polypeptide for use in medicine, the polypeptide comprising asequence given in SEQ ID NO: 1, 2, 3, 4, 5, 6 or 11 or a fragment of atleast 8 contigous amino acids of the SEQ ID NO: 1, 2, 3, 4, 5, 6 or 11sequences, and the polypeptide being capable of inducing a T cellresponse.
 2. A polypeptide for use in medicine according to claim 1;wherein the polypeptide is from 8 to 10 amino acids long.
 3. Apolypeptide for use in medicine according to claim 1; wherein thepolypeptide is from 12 to 25 amino acids long.
 4. A polypeptide for usein medicine according to any preceding claim; wherein the T cellresponse increases the number and/or activity of T helper and/or Tcytotoxic cells.
 5. A nucleic acid molecule for use in medicine; whereinthe nucleic acid molecule: a) has a strand that encodes a polypeptide asdescribed in any of claims 1 to 4; b) has a strand that is complementarywith a strand as described in a) above; or c) has a strand thathybridises with a molecule as described in a) or b) above (eg. understringent conditions).
 6. A vector or cell for use in medicinecomprising a nucleic acid molecule according to claim
 11. 7. A Tlymphocyte for use in medicine; wherein the T lymphocyte is capable ofkilling a cell expressing a polypeptide according to any of claims 1 to4 or of helping in the killing of such a cell.
 8. The use of apolypeptide as described in any of claims 1 to 4, a nucleic acid asdescribed in claim 5, or a vector or cell as described in claim 6, inthe preparation of a medicament for treating cancer, or in thepreparation of a diagnostic for diagnosing cancer.
 9. The use accordingto claim 8; wherein the cancer is mammalian cancer.
 10. The useaccording to claim 9; wherein the cancer is human cancer.
 11. The useaccording to any of claims 8 to 10; wherein the cancer is breast cancer,prostate cancer, pancreatic cancer, colo-rectal cancer, lung cancer,malignant melanoma, leukaemia, lymphoma, ovarian cancer, cervical canceror a biliary tract carcinoma.
 12. The use according to any of claims 8to 11; wherein the medicament is a vaccine.
 13. The use according to anyof claims 8 to 11; wherein the diagnostic is provided in a kit.
 14. Apharmaceutical composition comprising a polypeptide as described in anyof claims 1 to 4, a nucleic acid as described in claim 5, or a vector orcell as described in claim
 6. 15. A pharmaceutical composition accordingto claim 29 further comprising a polypeptide capable of inducing a Tcell response directed against a polypeptide produced by an oncogene oragainst a mutant tumour suppressor protein, or a nucleic acid encodingsuch a polypeptide.
 16. A pharmaceutical composition according to claim15; wherein said oncogene or mutant tumour suppressor protein isp21-ras, Rb, p53, abl, gip, gsp, ret or trk.
 17. A pharmaceuticalcomposition according to any of claims 14 to 16 comprising apharmaceutically acceptable carrier, diluent, additive, stabiliser,and/or adjuvant; said composition or combined preparation optionallyfurther including one or more of a cytokine or a growth factor.
 18. Apharmaceutical composition according to any of claims 14 to 17 which isa vaccine.
 19. A mixture of T lymphocytes for use in medicine comprisinga T helper cell as described in claim 17 or a clonal cell line of suchcells as described in claim 18 and a T cytotoxic cell as described inclaim 17 or a clonal cell line of such cells as described in claim 18.20. The use of a polypeptide as described in any of claims 1 to 10, anucleic acid as described in claim 11, a vector or cell as described inclaim 12, a binding agent as described in any of claims 13 to 15, a Tlymphocyte as described in claim 16 or 17, a cell line as described inclaim 18, or a mixture of T lymphocytes as described in claim 19, in thepreparation of a medicament for treating cancer, or in the preparationof a diagnostic for diagnosing cancer.
 21. The use according to claim20; wherein the cancer is mammalian cancer.
 22. The use according toclaim 21; wherein the cancer is human cancer.
 23. The use according toany of claims 20 to 22; wherein the cancer is breast cancer, prostatecancer, pancreatic cancer, colo-rectal cancer, lung cancer, malignantmelanoma, leukaemia, lymphoma, ovarian cancer, cervical cancer or abiliary tract carcinoma.
 24. The use according to any of claims 20 to23; wherein the medicament is a vaccine.
 25. The use according to any ofclaims 20 to 23; wherein the medicament is an antisense molecule or iscapable of generating an antisense molecule in vivo.
 26. The useaccording to any of claims 20 to 23; wherein the diagnostic is providedin a kit.
 27. The use according to claim 26; wherein the kit comprisesmeans for generating a detectable signal (eg. a fluorescent label, aradioactive label) or a detectable change (eg. an enzyme-catalysedchange).
 28. The use according to claim 26 or claim 27; wherein the kitincludes instructions for use in diagnosing cancer.
 29. A pharmaceuticalcomposition comprising a polypeptide as described in any of claims 1 to10, a nucleic acid as described in claim 11, a vector or cell asdescribed in claim 12, a binding agent as described in any of claims 13to 15, a T lymphocyte as described in claim 16 or 17, a cell line asdescribed in claim 18, or a mixture of T lymphocytes as described inclaim
 19. 30. A pharmaceutical composition according to claim 29 furthercomprising a polypeptide capable of inducing a T cell response directedagainst a polypeptide produced by an oncogene or against a mutant tumoursuppressor protein, or a nucleic acid encoding such a polypeptide, or abinding agent that binds such a polypeptide, or a T cell that is capableof killing a cell expressing such a polypeptide or of helping in thekilling of such a cell.
 31. A pharmaceutical composition according toclaim 30; wherein said oncogene or mutant tumour suppressor protein isp21-ras, Rb, p53, abl, gip, gsp, ret or trk.
 32. A combined preparationcomprising a component from claim 29 and a component from claim 30 orclaim 31 for simultaneous, separate or sequential use in anticancertherapy.
 33. A pharmaceutical composition according to any of claims 29to 31, or a combined preparation according to claim 32, comprising apharmaceutically acceptable carrier, diluent, additive, stabiliser,and/or adjuvant; said composition or combined preparation optionallyfurther including one or more of: a cytokine or growth factor (eg. IL-2,IL-12, and/or GM-CSF) and another polypeptide arising from a frameshiftmutation (eg. a frameshift mutation in the BAX or hTGFγ-RII gene.)
 34. Apharmaceutical composition according to any of claims 29 to 31 or 33, ora combined preparation according to claim 32 or 33, which is a vaccine.35. A pharmaceutical composition according to any of claims 29 to 31 or33, or a combined preparation according to claim 32 or 33, whichcomprises or is capable of producing antisense molecules.
 36. Adiagnostic composition comprising a polypeptide as described in any ofclaims 1 to 10, a nucleic acid as described in claim 11, a vector orcell as described in claim 12, a binding agent as described in any ofclaims 13 to 15, a T lymphocyte as described in claim 16 or 17, a cellline as described in claim 18, or a mixture of T lymphocytes asdescribed in claim
 19. 37. A diagnostic kit as described in any ofclaims 26 to
 28. 38. A polypeptide as described in any of claims 1 to10, optionally in isolated form; wherein the polypeptide is not apolypeptide consisting of the sequences shown in FIG.
 4. 39. A nucleicacid as described in claim 11, optionally in isolated form; wherein thenucleic acid is not a nucleic acid encoding the polypeptide excludedfrom claim 38 and is also not a nucleic acid as shown in FIG. 4 or FIG.5.
 40. A vector or cell as described in claim 12, optionally in isolatedform.
 41. A binding agent as described in any of claims 13 to 15,optionally in isolated form.
 42. A T lymphocyte as described in claim 16or 17, optionally in isolated form.
 43. A clonal cell line as describedin claim 18, optionally in isolated form.
 44. A mixture of T lymphocytesas described in claim
 19. 45. A machine readable data carrier (eg. adisk) comprising the sequence of a polypeptide according to claim 38 orof a nucleic acid according to claim
 39. 46. A method comprising usingthe sequence of a polypeptide according to claim 38 or a nucleic acidmolecule according to claim 39 to perform sequence identity studies,sequence homology studies, or hybridisation studies.
 47. A methodcomprising using the sequence of a polypeptide according to claim 38 ora nucleic acid molecule according to claim 39 to predict structureand/or function (eg. to predict anti-cancer activity).
 48. The use of amethod according to claim 46 or claim 47 in a drug development orscreening procedure.
 49. A drug identified or selected by a procedure asdescribed in claim
 48. 50. A computer or database that displays orstores a sequence of a polypeptide according to claim 38 or a nucleicacid molecule according to claim 39 or that is set up to perform amethod according to claim 46 or
 47. 51. The invention as substantiallyhereinbefore described with reference to the accompanying figures andexamples.